Various hydroxylated estrogens are major metabolites of estrogens in man and animals, including the 2-hydroxylated catechol estrogens. Our previous work with catechols, such as gamma-methyldopa, showed that they could be oxidized to electrophilic derivatives which bind to tissue macromolecules. We, therefore, investigated the possibility that estrogens and catechol estrogens might be activated in a simillr manner. 2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17 gamma-ethynylestradiol were found to be converted by rat liver microsomes to reactive metabolites that become covalently bound to microsomal protein. The covalent binding required microsomes, oxygen, and NADPH. The NADPH could be replaced by xanthine-xanthine oxidase which is known to generate superoxide anions. The binding was substantially inhibited by superoxide dismutase, 30 per cent in those incubations containing NADPH and 98 per cent in those containing the xanthine-xanthine oxidase system. The binding was inhibited 70 per cent in an atmosphere of Co:O2 (9:1). These experiments plus other studies using various inhibitors and inducers of the P-450 system, are consistent with a mechanism for the covalent binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion. Studies are now in progress to determine the significance of these experiments to various kinds of estrogen toxicity which have recently been documented.